According to the confocal result, we found that Plan A doesn’t work well. Therefore, we decided to use Plan B only.
(1) Nanoparticle solution preparation
250μl of nanoparticle stock solution (conc.=40mg/ml) is diluted with 1750μl HBSS (Concentration: 5mg/ml)
Solution with concentration 5 times as working concentration is prepared here.
Total volume 500μl for each concentration
(2) SiRNA containing solution preparation:<br/> For each well, 300μl of nanoparticle solution is needed for treatment.
For SiRNA containing nanoparticle solution
60μl is added to 240 μl serum free medium to dilute 5 times.
Total volume: 60x 2(duplicates) x2(with magnetic field/without magnetic field)=240μl
Take in account the pipetting error, 240μl -> 320μl for each concentration solution containing nanoparticles
SiRNA stock concentration:40μM
SiRNA concentration needed for treatment 200nM
SiRNA concentration for nanoparticle solution preparation: 1000nM
( *Remarks:The final concentration of SiRNA containing nanoparticle solution is not calculated here.)
Remarks: if under this condition, the nanoparticle is too concentrated for conjugation.
For SiRNA containing incomplete medium solution (experiment 9,10)
Volume of SiRNA containing medium
= 60μlx2 (duplicates) x 2(experiment 9,10)
Take in account of pipetting error, 240μl-> 320μl
After adding SiRNA to both nanoparticle and HBSS solution, the tubesare stored in 4 centigrade refrigerator to allow conjugating overnight for 24hrs.
(1)Precoat the 4 well plate
(2) Seeding cells
After cell count, calculate the volume should be added into each well according to the cell number of 5x104 cells/well.
Place in incubator overnight to allow cell growth